Nucleoside triphosphate and cation requirement for dopamine uptake by plain synaptic vesicles isolated from rat...

Plain synaptic vesicles were partially purified from rat cerebrums and their [3H]dopamine uptake was investigated. The addition of MgCl2 plus ATP and of CaCl2 plus ATP increased the uptake 16 times and 5.5 times, respectively, higher than the control level, whereas the separate addition of these agents augmented the level at most 2.5 times. GTP stimulated the uptake as well as ATP, whereas UTP was one-third as effective and CTP, ADP, and AMP were all ineffective. Adenylyl imidodiphosphate was not only ineffective, but strongly reduced the ATP-dependent uptake. The half-maximal level of the ATP-dependent uptake was reached within 1.5 min after the start of incubation at 25 degrees C. The incorporation peaked at 5-10 min, then gradually declined to half-maximum at 45 min. This decline was prevented by the further addition of small amounts of CaCl2. There was no uptake at 0 degrees C, and the incorporation rate was at least 2 times faster at 37 degrees C than it was at 25 degrees C. The apparent Michaelis constant for [3H] dopamine was 1.6 muM. The half maximal inhibition of uptake was obtained at 0.1 muM reserpine; neither colchicine nor ouabain showed significant inhibition. The ATP-dependent uptake was not affected by K+, Na+, and Cl-, but was drastically decreased by isotonic phosphate buffer.